Cultural medium for maintaining neural cells in ambient atmosphere

ABSTRACT

The present invention provides a minimal essential medium for maintaining neural cell or tissue viability in an environment containing ambient levels of CO 2 . The medium contains less than about 2000 μM bicarbonate, a buffer having a pKa of from about 6.9 to about 7.7, wherein the medium is free of ferrous sulfate, glutamate and aspartate, from 0 to about 3000 μM CaCl 2 , from about 0.05 to about 0.8 μM Fe(NO 3 ) 3 , from about 2500 to about 10000 μM KCl, from 0 to about 4000 μM MgCl 2 , from about 74000 to about 103000 μM NaCl, from about 400 to about 2000 μM NaHCO 3 , from about 250 to about 4000 μM NaH 2 PO 4 , from about 0.2 to about 2 μM ZnSO 4 , from about 2500 to about 50000 μM glucose and from about 23 to about 500 μM sodium pyruvate. The present invention also provides a process of extending neural cell or tissue viability in an atmosphere having ambient levels of carbon dioxide whereby the neural cells or tissue are placed in such a medium. Preferably, the medium is supplemented with a growth-promoting medium that contains effective amounts of hormones, essential fatty acids and anti-oxidants for neural cells.

This application is a 371 of PCT/US97/05875, filed Apr. 9, 1997, whichclaims priority to U.S. Provisional application Serial No. 60/015,049,filed Apr. 9, 1996.

TECHNICAL FIELD OF THE INVENTION

The field of this invention is cell culture media. More particularly,the present invention pertains to a medium for neural cells or tissuethat maintains viability of those cells or tissue in an atmospherehaving ambient levels of carbon dioxide.

BACKGROUND OF THE INVENTION

A major problem attendant to studies of central nervous system tissue isthe maintenance of cell viability of such tissues. The inability tomaintain central nervous system tissue viability in culture forprolonged periods of time and under various environmental conditions hasimpeded the development of effective therapeutic regimens for treatingcentral nervous system disorders.

A nutrient balanced salt solution (medium) for maintaining centralnervous system tissue viability in a high-carbon dioxide atmosphere (5%CO₂) has recently been developed. That medium, Neurobasal™ (Gibco/LifeTechnologies, Inc., Gaithersburg, Md.), is a bicarbonate buffered mediumoptimized for the growth of embryonic rat hippocampal neurons at a pH of7.3 in 5% CO₂. Neurobasal™ is a derivative of Dulbecco's ModifiedEagle's Medium (DMEM) and was formulated to optimize embryonic rathippocampal cell survival. When compared to DMEM, Neurobasal™ has lessNaCl and less NaHCO₃, resulting in a lower osmolality, and lesseramounts of cysteine and glutamine, resulting in diminished glial growth.In addition, Neurobasal™ contains alanine, asparagine, proline andvitamin B12, all of which are absent from DMEM.

Although neurons can be maintained in a 5% CO₂ atmosphere in this highbicarbonate medium, when supplemented with B27 (a hormone andanti-oxidant supplement available from Life Technologies, Inc.), neuronsundergo rapid death when transferred to ambient CO₂ (0.2%) conditions.Death is associated with a rapid rise in medium pH to a value of 8.1.

The preparation and study of neural tissue and cells frequently requiresthe use of ambient CO₂ levels outside of an incubator. Existing methodsfor controlling the pH of cells outside of incubators include the use ofweak buffers (e.g., as found in Dulbecco's modified Eagle's medium or L15 medium) and the use of continuous gassing with 5-10% CO₂ to maintainphysiological pH.

A simple test, however, shows that ambient CO₂ causes the pH of DMEM toquickly rise to a value of 8.1 outside the incubator. The commonpractice of buffering with HEPES slows but does not prevent thissubstantial alkalinization. The practice of continuously gassing tissuesto maintain high CO₂ levels and physiological pH is cumbersome andexpensive. There continues to be a need in the art, therefore, for amedium that can maintain physiological pH and neural cell viability inambient CO₂ conditions.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a minimal essential mediumfor maintaining neural cell or tissue viability in an environmentcontaining ambient levels of CO₂. The medium contains less than about2000 μM bicarbonate, has an osmolality of from about 230 mOsm to about300 mOsm, contains a buffer having a pKa of from about 6.9 to about 7.7,and is free of ferrous sulfate, glutamate and aspartate. A medium of thepresent invention is effective in maintaining viability of neural cellsor tissue derived from embryonic tissues or adult tissues.

Where the cells or tissue are of embryonic origin, the osmolality isfrom about 230 mOsm to about 250 mOsm. Such a medium comprises, in finalconcentration, 0 to about 3000 μM CaCl₂, 0.05 to about 0.8 μM Fe(NO₃)₃,2500 to about 10000 μM KCl, 0 to about 4000 μM MgCl₂, 74000 to about81000 μM NaCl, 400 to about 2000 μM NaHCO₃, 250 to about 4000 μMNaH₂PO₄, 0.2 to about 2 μM ZnSO₄, 2500 to about 5000 μM glucose, 0 toabout 100 μM phenol red, and 23 to about 500 μM sodium pyruvate.

Where the neural cells or tissue are of adult origin, the osmolality isfrom about 250 mOsm to about 300 mOsm. Such a medium comprises, in finalconcentration, 0 to about 3000 μM CaCl₂, 0.05 to about 0.8 μM Fe(NO₃)₃,2500 to about 10000 μM KCl, 0 to about 4000 μM MgCl₂, 86000 to about103000 μM NaCl, 400 to about 2000 μM NaHCO₃, 250 to about 4000 μMNaH₂PO₄, 0.2 to about 2 μM ZnSO₄, 2500 to about 5000 μM glucose, 0 toabout 100 μM phenol red, and 23 to about 500 μM sodium pyruvate.

A preferred buffer is 3-[N-morpholino]propane-sulfonic acid (MOPS). Amedium of the present invention contains effective amo unts of at leastten essential amino acids. In one embodiment, the medium contains, infinal concentration: a) from about 250 to about 2500 μM each ofL-isoleuoine, L-leucine, L-threonine and L-valine; b) from about 150 toabout 1500 μM L-glutamine; c) from about 120 to about 1200 μM each ofL-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) fromabout 60 to about 600 μM each of L-histidine and L-methionine; e) fromabout 25 to about 250 μM L-tryptophan; f) from about 25 to about 250 μML-proline; g) from about 6 to about 60 μM L-alanine; h) from about 3 toabout 30 μM L-cysteine; and i) from about 1.5 to about 15 μM each ofL-asparagine and L-lysine.

A medium of the present invention further includes vitamins in amountseffective to sustain neural cell or tissue viability. The mediumcontains, in final concentration, from about 12 to about 120 μMi-inositol, from about 10 to about 100 μM niacinamide, from about 9 toabout 90 μM choline chloride, from about 6 to about 60 μM pyridoxal,from about 3 to about 30 μM thiamine, from about 2.5 to about 25 μM eachof folic acid and D-Ca pantothenate, from about 0.3 to about 3 μMriboflavin, and from about 0.06 to about 0.6 μM vitamin B12.

In another aspect, the present invention provides a process of extendingneural cell or tissue viability in an atmosphere having ambient levelsof carbon dioxide. The process includes the steps of placing neuralcells or tissue in a medium of the present invention and maintaining thecells or tissue in that medium under ambient CO₂ conditions. In apreferred embodiment of a process of the present invention, the mediumis supplemented with a serum-free growth promoting supplement thatcontains effective amounts of hormones, essential fatty acids andanti-oxidants for neural cells. A preferred growth-promoting supplementcontains biotin, L-carnitine, corticosterone, ethanolamine,D(+)-galactose, reduced glutathione, linoleic acid, linolenic acid,progesterone, putrescine, retinyl acetate, selenium, triodo-1-thyronine,DL-α tocopherol, DL-α tocopherol acetate, bovine albumin, catalase,insulin, superoxide dismutase and transferrin.

The present invention also provides a composition comprising neuralcells or tissue in a medium as set forth above. The medium containingthe cells or tissue can optionally be supplemented with agrowth-promoting supplement as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, which form a portion of the specification:

FIG. 1 shows the viability of neurons cultured for four days inB27-supplemented Neurobasal™ in 5% CO₂ and then transferred to a mediumof the present invention either alone (-◯-) or supplemented withB27(--).

FIG. 2 shows survival of hippocampal neurons dissected from the brainand placed in either a medium of the present invention supplemented withB27(-◯-) or L-15/B27(--) at ambient CO₂ levels. After storage for theindicated period at 8° C., individual cells were isolated and placed inB27-supplemented Neurobasal™ at 5% CO₂for four days.

DETAILED DESCRIPTION OF THE INVENTION

I. Composition of Matter

In one aspect, the present invention provides a medium for neural cellsor tissue maintained in an environment containing ambient amounts ofcarbon dioxide (CO₂). As is well known in the art, ambient air containsless than about 0.25% CO₂ and, more typically, about 0.2% CO₂. A mediumof the present invention is designed and formulated to allow forprolonged survival and viability of neural cells or tissue exposed tosuch low CO₂ levels.

As is well known in the art, there are a variety of preparative steps toobtain primary neural cell cultures. By way of example, specific regionsof the brain are typically dissected as a tissue and placed in asuitable medium. This neural tissue is further processed to obtain cellswhich are typically plated in a culture container and the medium isplaced over the plated tissue. In both cases, the medium is changed asneeded to maintain viability. As used herein, the terms “suspended” or“cultured” are used to refer both to suspensions of tissue, orindividual cells and plated individual cells. As used herein, the phrase“neural cells” means either individual cells of the same or differenttype whether those cells exist as isolated cells or exist in the form ofaggregates or collections.

A medium of the present invention is a derivative of a basal mediumpreviously described by the present inventor to provide prolongedviability of neural cells or tissue in a high-CO₂ environment (e.g., 5%CO₂) such as typically used in cell incubators (Brewer et al., J.Neuroscience Res., 35:567-576, 1993). The basal medium described in thatpublication was designated Neurobasal™.

A medium of the present invention contains the same nutrients (e.g.,glucose, amino acids) and vitamins as Neurobasal™. Differences betweenNeurobasal™ and a medium of the present invention are changes in thelevels of bicarbonate and sodium chloride and the use of MOPS as theinert buffer (HEPES is used in Neurobasal™).

Like Neurobasal™, a medium of the present invention can be used withneural cells or tissue derived from any area of the central nervoussystem of a mammal, including humans. The cells or tissues can bederived or obtained from embryonic, neo-natal or adult central nervoussystem tissue. A medium of the present invention can also be used tomaintain viability of neural cells or tissue obtained after death of thedonor animal (post-mortem harvesting). Post-mortem tissue harvesting isparticularly important where the donor animal is killed rather thananesthetized prior to tissue extraction.

A medium of the present invention is characterized by the presence ofvery low levels of a bicarbonate buffer system. The pH value of themedium is controlled predominantly by non-bicarbonate buffers. Thosebuffers include sodium phosphate (e.g., NaH₂PO₄) and inert buffershaving a pKa between a pH value of about 6.9 and about 7.7. Such inertbuffers are well known to those of skill in the art and readilyavailable from commercial sources. A preferred such inert buffer is3-[N-morpholino] propane-sulfonic acid (MOPS) having a pKa of about 7.2.An inert buffer used in a medium of the present invention must benon-toxic toward neural cells. Thus, for example, the use of HEPES,which has been shown to be phototoxic to certain neural cells should beavoided. The inert buffer is present at a final concentration of fromabout 5000 μM to about 25000 μM and, more preferably from, about 8000 μMto about 12000 μM.

A medium of the present invention is effective in maintaining viabilityof neural cells or tissue derived from embryonic tissues or adulttissues. Where the cells or tissue are of embryonic origin, the mediumhas an osmolality from about 230 mOsm to about 250 mOsm. Such a mediumcomprises, in final concentration, from 0 to about 3000 μM CaCl₂, fromabout 0.05 to about 0.8 μM Fe(NO₃)₃, from about 2500 to about 10000 μMKCl, from 0 to about 4000 μM MgCl₂, from about 74000 to about 81000 μMNaCl, from about 400 to about 2000 μM NaHCO₃, from about 250 to about4000 μM NaH₂PO₄, from about 0.2 to about 2 μM ZnSO₄, from about 2500 toabout 50000 μM glucose, from 0 to about 100 μM phenol red, and fromabout 23 to about 500 μM sodium pyruvate. More preferably, such a mediumcomprises, in final concentration, from about 900 to about 2500 μMCaCl₂, from about 0.1 to about 0.4 μM Fe(NO₃)₃, from about 4000 to about7000 μM KCl, from 500 to about 1500 μM MgCl₂, from about 75000 to about77000 μM NaCl, from about 600 to about 1200 μM NaHCO₃, from about 600 toabout 1200 μM NaH₂PO₄, from about 0.4 to about 1.2 μM ZnSO₄, from about15000 to about 35000 μM glucose, from about 15 to about 40 μM phenolred, and from about 150 to about 250 μM sodium pyruvate.

An especially preferred such medium comprises, in final concentration,1800 μM CaCl₂, 0.2 μM Fe(NO₃)₃, 5360 μM KCl, 812 μM MgCl₂, 76000 μMNaCl, 880 μM NaHCO₃, 900 μM NaH₂PO₄, 0.67 μM ZnSO₄, 25000 μM glucose, 23μM phenol red, and 230 μM sodium pyruvate.

Where the neural cells or tissue are of adult origin, the osmolality isis from about 250 mOsm to about 300 mOsm. Such a medium comprises, infinal concentration, from 0 to about 3000 μM CaCl₂, from about 0.05 toabout 0.8 μM Fe(NO₃)₃, from about 2500 to about 10000 μM KCl, from 0 toabout 4000 μM MgCl₂, from about 86000 to about 103000 μM NaCl, fromabout 400 to about 2000 μM NaHCO₃, from about 250 to about 4000 μMNaH₂PO₄, from about 0.2 to about 2 μM ZnSO₄, from about 2500 to about50000 μM glucose, from 0 to about 100 μM phenol red, and from about 23to about 500 μM sodium pyruvate. More preferably, such a mediumcomprises, in final concentration, from about 900 to about 2500 μMCaCl₂, from about 0.1 to about 0.4 μM Fe(NO₃)₃, from about 4000 to about7000 μM KCl, from 500 to about 1500 μM MgCl₂, from about 83000 to about95000 μM NaCl, from about 600 to about 1200 μM NaHCO₃, from about 600 toabout 1200 μM NaH₂PO₄, from about 0.4 to about 1.2 μM ZnSO₄, from about15000 to about 35000 μM glucose, from about 15 to about 40 μM phenolred, and from about 150 to about 250 μM sodium pyruvate.

An especially preferred such medium comprises, in final concentration,1800 μM CaCl₂, 0.2 μM Fe(NO₃)₃, 5360 μM KCl, 812 μM MgCl₂, 89000 μMNaCl, 880 μM NaHCO₃, 900 μM NaH₂PO₄, and 0.67 μM ZnSO₄, 25000 μMglucose, 23 μM phenol red, and 230 μM sodium pyruvate.

A medium of the present invention contains effective amounts of at leastten essential amino acids. In one embodiment, the medium contains, infinal concentration: a) from about 250 to about 2500 μM each ofL-isoleucine, L-leucine, L-threonine and L-valine; b) from about 150 toabout 1500 μM L-glutamine; c) from about 120 to about 1200 μM each ofL-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) fromabout 60 to about 600 μM each of L-histidine and L-methionine; e) fromabout 25 to about 250 μM L-tryptophan; f) from about 25 to about 250 μML-proline; g) from about 6 to about 60 μM L-alanine; h) from about 3 toabout 30 μM L-cysteine; and i) from about 1.5 to about 15 μM each ofL-asparagine and L-lysine. More preferably, the medium contains, infinal concentration: a) from about 500 to about 1200 μM each ofL-isoleucine, L-leucine, L-threonine and L-valine; b) from about 250 toabout 750 μM L-glutamine; c) from about 200 to about 600 μM each ofL-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) fromabout 100 to about 300 μM each of L-histidine and L-methionine; e) fromabout 50 to about 125 μM L-tryptophan; f) from about 55 to about 90 μML-proline; g) from about 15 to about 30 μM L-alanine; h) from about 7 toabout 15 μM L-cysteine; and i) from about 2.5 to about 7.5 μM each ofL-asparagine and L-lysine.

In a most preferred embodiment, the medium contains, in finalconcentration: a) 800 μM each of L-isoleucine, L-leucine, L-threonineand L-valine; b) 500 μM L-glutamine; c) 400 μM each of L-arginine,glycine, L-phenylalanine, L-serine and L-tyrosine; d) 200 μM each ofL-histidine and L-methionine; e) 80 μM L-tryptophan; f) 67 μM L-proline;g) 20 μM L-alanine; h) 10 μM L-cysteine; and i) 5 μM each ofL-asparagine and L-lysine.

A medium of the present invention further includes vitamins in amountseffective to sustain neural cell or tissue viability. The mediumcontains, in final concentration, from about 12 to about 120 μMi-inositol, from about 10 to about 100 μM niacinamide, from about 9 toabout 90 μM choline chloride, from about 6 to about 60 μM pyridoxal,from about 3 to about 30 μM thiamine, from about 2.5 to about 25 μM eachof folic acid and D-Ca pantothenate, from about 0.3 to about 3 μMriboflavin, and from about 0.06 to about 0.6 μM vitamin B12. Morepreferably, the final concentration of vitamins is from about 20 toabout 60 μM i-inositol, from about 15 to about 50 μM niacinamide, fromabout 20 to about 40 μM choline chloride, from about 10 to about 30 μMpyridoxal, from about 5 to about 15 μM thiamine, from about 5 to about12 μM each of folic acid and D-Ca pantothenate, from about 0.5 to about1.5 μM riboflavin, and from about 0.1 to about 0.3 μM vitamin B12.

In an especially preferred embodiment, the medium contains, in finalconcentration, 40 μM i-inositol, 30 μM niacinamide, 28 μM cholinechloride, 20 μM pyridoxal, 10 μM thiamine, 8 μM each of folic acid andD-Ca pantothenate, 1 μM riboflavin, and 0.2 μM vitamin B12.

II. Process of Maintaining Neural Cell Viability

A medium of the present invention has many uses, all of which arerelated to the ability of that medium to prolong survival of neuralcells or tissues in a low CO₂ environment.

In one embodiment, the medium can be used to store neural cells ortissue. The cells, once obtained from any brain region, are placed in amedium and maintained under ambient CO₂ conditions. By way of example,tissue from the central nervous system can be placed in a medium of thepresent invention and stored at reduced temperature (e.g., 2° C. to 15°C.) The storage of neural cells or tissue is useful both in preparingsuch cells for experimental procedures and for maintaining cells priorto transplantation or implantation for therapeutic purposes. Exemplarysuch uses include ex vivo gene therapy, repair of damaged circuits,clonal expression and proliferating autologous or heterologous cells fortransplant.

A medium of the present invention can also be used to maintain theviability of plated neural cells that are undergoing study ormanipulation. Thus, for example, cells can be maintained under normalambient conditions during electrophysiological examination. In a similarfashion, neural cells or tissue placed in a medium of the presentinvention can be transfected or transformed with expression vectorscarrying gene inserts. Once transformed, those cells can be implantedinto various regions of the brain as part of a gene therapy regimen. Amedium of the present invention can also be used as a perfusate orirrigant for central nervous system tissue during central nervous systemoperations.

Use of a medium of the present invention has the advantage that neuralcells or tissue can be studied or manipulated under normal, ambientconditions without exposing those cells or tissue to the deleteriouseffects of high CO₂ levels and changes in pH. Still further, there is noneed to continuously gas a medium of the present invention. Thus, use ofa medium of the present invention results in substantial time and costsavings.

When used to sustain or prolong neural cell or tissue viability, amedium of the present invention is preferably supplemented with agrowth-promoting supplement that contains essential fatty acids,hormones and anti-oxidants needed for growth of the particular neuralcells being cultured. Such growth-promoting media are well known in theart. By way of example, the present inventor has formulated agrowth-promoting media for use with embryonic rat hippocampal cells.That medium is designated B27 and is commercially available fromGIBCO/Life Technologies, Inc., Gaithersburg, Md. The growth promotingsupplement, B27, contains essential fatty acids, hormones andanti-oxidants in amounts that optimize embryonic hippocampal neurongrowth. The fatty acids, hormones and anti-oxidants in B27 are biotin,L-carnitine, corticosterone, ethanolamine, D(+)-galactose, reducedglutathione, linoleic acid, linolenic acid, progesterone, putrescine,retinyl acetate, selenium, triodo-1-thyronine, DL-α tocopherol, DL-αtocopherol acetate, bovine albumin, catalase, insulin, superoxidedismutase and transferrin.

The present invention also provides a composition comprising neuralcells or tissue placed in a medium as set forth above. The culture canfurther comprise a growth-promoting supplement as set forth above.

The following Example illustrates preferred embodiments of the presentinvention and is not limiting of the specification and claims in anyway.

EXAMPLE 1

Two basic types of experiments were conducted. In the first, ratembryonic day 18 hippocampal neurons were isolated and cultured aspreviously described at 160 cells/mm² in B27-supplemented Neurobasal™(Life Technologies, Inc., Gaithersburg, Md.) on 24-well plasticsubstrates coated with polylysine. After 4 days in culture at 37° C. in9% O₂, 5% CO₂, the entire medium was changed to a pre-warmed CO₂independent medium supplemented with B27 as indicated. The compositionof that CO₂ independent medium is set forth below in Table 1.

TABLE 1 Component Concentration in μM inorganic salts CaCl₂ (anhydrous)1800 Fe(NO₃)₃ 9H₂O 0.2 KCl 5360 MgCl₂ (anhydrous) 812 NaCl 76000 NaHCO₃880 NaH₂PO₄H₂O 900 ZnSO₄7H₂O 0.67 other components D-glucose 25000phenol red 23 MOPS 10000 sodium pyruvate 230 amino acids L-alanine 20L-arginine HCl 400 L-asparagine H₂O 5 L-cysteine 10 L-glutamine 500glycine 400 L-histidine HCl H₂O 200 L-isoleucine 800 L-leucine 800L-lysine HCl 5 L-methionine 200 L-phenylalanine 400 L-proline 67L-serine 400 L-threonine 800 L-tryptophan 80 L-tyrosine 400 L-valine 800vitamins D-Ca pantothenate 8 choline chloride 28 folic acid 8 i-inositol40 niacinamide 30 pyridoxal HCl 20 riboflavin 1 thiamine HCl 10 vitaminB12 0.2

Culture was continued at 37° C. in a bacteriologic incubator at ambientO₂ and CO₂. The plates were placed in a Plexiglas plastic box adjacentto open dishes of water for hydration. At various times, plates wereremoved for counting live cells by staining with fluorescein diacetateand dead cells with propidium iodide. Survival was calculated as thenumber of live cells divided by the total cells (live+dead).

The data in FIG. 1 show that HEPES, bicarbonate buffered Neurobasal™supplemented with B27 produced rapid loss of neuron viability in ambientCO₂, with an LD₅₀ of less than 6 hr. When this medium was moreappropriately buffered and the bicarbonate concentration reduced, muchbetter survival was obtained. This new formulation (See Table 1)extended the LD₅₀ beyond 24 hr. Slightly inferior results were obtainedwith a simple balanced salt solution (Hank's, Life Technologies, Inc.#14060-016) supplemented with 0.1% glucose, 1 μM sodium bicarbonate, 1μM pyruvate, 10 μM HEPES, pH 7.3. Further improvement in viability wasobtained with the use of anti-oxidants, essential fatty acids, hormonesand other ingredients as in the B27 supplement. With the B27 supplement,full viability was maintained for at least 2 days in culture in ambientCO₂. The LDSO was extended beyond 3 days. Developed axons and dendritesshowed little evidence of neurodegeneration such as retracted or beadedneurites or swollen somae. These neurons after 3 days in ambient CO₂were comparable to neurons cultured in 5% C₂.

Comparisons of viability and sprouting were also made for ambient and 5%CO₂conditions after one and four days in culture. After one day, viablecells in cultures started in 5% CO₂ in B27-supplemented Neurobasal™ were63±10% of those plated (mean±S.D., n=12) and 54±9% of these had neuritesprouts greater than one cell diameter. In ambient CO₂ and theB27-supplemented medium of Table 1, viability on day 1 was reduced to50±7% (p<0.05) and sprouting was reduced to 22±12% (p<0.001). After 4days, all cells were dead in the B27-supplemented medium of Table 1 inambient CO₂ while normal culture in B27-supplemented Neurobasal™ in 5%CO₂ produced 71% viability. These data show that hippocampal neuronsneed a start in 5% CO₂ to remain healthy.

In the second type of experiment, hippocampi were dissected as usual,but placed in 2 ml of the medium of Table 1 supplemented with B27 in a15 ml centrifuge tube (Corning, Oneonta, N.Y.). The tube was placed in arefrigerator at 8° C. At various times, the tube was removed to alaminar flow hood at room temperature. One ml of the medium was removedand saved. The hippocampus in 1 ml medium was triturated by mild suctionand expulsion 10 times (or until most pieces of tissue are dispersed)through the plastic tip of a 1 ml pipettor (Gilson, Middleton, Wis.).The volume was returned to 2 ml with the original medium. After lettingundispersed pieces settle for 3 min., the supernatant was transferredand centrifuged for 1 min. at 200×G. The supernatant was discarded. Toloosen the pellet, the tube was agitated by hand before resuspension in1 ml of B27-supplemented Neurobasal™ medium. After counting cells thatexcluded 0.2% trypan blue, cells were plated and cultured as above.

The data in FIG. 2 show that hippocampal tissue can be stored for atleast one month in the refrigerator before losing half of the survivingcells, measured after 4 days in normal culture at 5% CO₂. These resultsare superior to prior studies reporting good survival after one week ofstorage, but little, if any, survival after 4 weeks in a low sodium, lowcalcium medium (Kawamoto et al, Brain Res., 384:84-93, 1986). Anothermedium sometimes used for trituration and storage is Leibovitz's L-15, amedium with high concentrations of amino acids (Leibovitz, Amr. J. Hyg.,78:173-180, 1963). The data show that the use of a medium of the presentinvention is superior to L-15 for refrigerated viable storage ofhippocampal tissue. The yield of viable cells after 1 week of tissuehibernation in B27-supplemented medium of Table 1 was 1.3±0.3 millioncells/hippocampus (mean±S.E., n=6). This is comparable to cell yieldsfrom fresh tissue.

What is claimed is:
 1. A medium for maintaining neural cell or tissueviability in an environment containing less than about 0.25% CO₂, themedium comprising: a) less than about 2000 μM bicarbonate; and b) abuffer having a pKa of from about 6.9 to about 7.7, wherein the mediumis free of ferrous sulfate, glutamate and aspartate; and c) from 0 toabout 3000 μM CaCl₂, from about 0.05 to about 0.8 μM Fe(NO₃)₃, fromabout 2500 to about 10000 μM KCl, from 0 to about 4000 μM MgCl₂, fromabout 74000 to about 103000 μM NaCl, from about 400 to about 2000 μMNaHCO₃, from about 250 to about 4000 μM NaH₂PO₄, from about 0.2 to about2 μM ZnSO₄, from about 2500 to about 50000 μM glucose and from about 23to about 500 μM sodium pyruvate.
 2. The medium according to claim 1further comprising from about 0 to about 100 μM phenol red.
 3. Themedium according to claim 1 wherein the neural cells or tissue are ofembryonic origin, and wherein the medium has an osmolality from about210 mOsm to about 250 mOsm.
 4. The medium according to claim 1 whereinthe neural cells or tissue are of adult origin, and wherein the mediumhas an osmolality is from about 250 mOsm to about 300 mOsm.
 5. Themedium according to claim 4 having a bicarbonate concentration of fromabout 600 μM to about 1200 μM.
 6. The medium according to claim 1wherein the buffer is (3-[N-morpholino]propanesulfonic acid).
 7. Themedium according to claim 6 wherein (3-[N-morpholino]propanesulfonicacid) is present in a concentration of from about 5000 to about 25000μM.
 8. The medium of claim 1 comprising from about 74000 to about 81000μM NaCl.
 9. The medium of claim 1 comprising from about 900 to about2500 μM CaCl₂, from about 0.1 to about 0.4 μM Fe(NO₃)₃ from about 4000to about 7000 μM KCl, from 500 to about 1500 μM MgCl₂, from about 75000to about 77000 μM NaCl, from about 600 to about 1200 μM NaHCO₃, fromabout 600 to about 1200 μM NaH₂PO₄, from about 0.4 to about 1.2 μMZnSO₄, from about 15000 to about 35000 μM glucose, and from about 150 toabout 250 μM sodium pyruvate.
 10. The medium of claim 9 furthercomprising from about 15 to about 40 μM phenol red.
 11. The medium ofclaim 10 comprising 1800 μM CaCl₂, 0.2 μM Fe(NO₃)₃, 5360 μM KCl, 812 μMMgCl₂, 76000 μM NaCl, 880 μM NaHCO₃ 900 μM NaH₂PO₄, 0.67 μM ZnSO₄, 25000μM glucose, 230 μM sodium pyruvate and 23 μM phenol red.
 12. The mediumaccording to claim 1 comprising a) from about 250 to about 2500 μM eachof L-isoleucine, L-leucine, L-threonine and L-valine; b) from about 150to about 1500 μM L-glutamine; c) from about 120 to about 1200 μM each ofL-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine; d) fromabout 60 to about 600 μM each of L-histidine and L-methionine; e) fromabout 25 to about 250 μM L-tryptophan; f) from about 25 to about 250 μML-proline; g) from about 6 to about 60 μM L-alanine; h) from about 3 toabout 30 μM L-cysteine; and i) from about 1.5 to about 15 μM each ofL-asparagine and L-lysine.
 13. The medium of claim 12 comprising a) fromabout 500 to about 1200 μM each of L-isoleucine, L-leucine, L-threonineand L-valine; b) from about 250 to about 750 μM L-glutamine; c) fromabout 200 to about 600 μM each of L-arginine, glycine, L-phenylalanine,L-serine and L-tyrosine; d) from about 100 to about 300 μM each ofL-histidine and L-methionine; e) from about 50 to about 125 μML-tryptophan; f) from about 55 to about 90 μM L-proline; g) from about15 to about 30 μM L-alanine; h) from about 7 to about 15 μM L-cysteine;and i) from about 2.5 to about 7.5 μM each of L-asparagine and L-lysine.14. The medium of claim 13 comprising a) 800 μM each of L-isoleucine,L-leucine, L-threonine and L-valine; b) 500 μM L-glutamine; c) 400 μMeach of L-arginine, glycine, L-phenylalanine, L-serine and L-tyrosine;d) 200 μM each of L-histidine and L-methionine; e) 80 μM L-tryptophan;f) 67 μM L-proline; g) 20 μM L-alanine; h) 10 μM L-cysteine; and i) 5 μMeach of L-asparagine and L-lysine.
 15. The medium according to claim 1comprising from about 12 to about 120 μM i-inositol, from about 10 toabout 100 μM niacinamide, from about 9 to about 90 μM choline chloride,from about 6 to about 60 μM pyridoxal, from about 3 to about 30 μMthiamine, from about 2.5 to about 25 μM each of folic acid and D-Capantothenate, from about 0.3 to about 3 μM riboflavin, and from about0.06 to about 0.6 μM vitamin B12.
 16. The medium of claim 15 comprisingfrom about 20 to about 60 μM i-inositol, from about 15 to about 50 μMniacinamide, from about 20 to about 40 μM choline chloride, from about10 to about 30 μM pyridoxal, from about 5 to about 15 μM thiamine, fromabout 5 to about 12 μM each of folic acid and D-Ca pantothenate, fromabout 0.5 to about 1.5 μM riboflavin, and from about 0.1 to about 0.3 μMvitamin B12.
 17. The medium of claim 16 comprising 40 μM i-inositol, 30μM niacinamide, 28 μM choline chloride, 20 μM pyridoxal, 10 μM thiamine,8 μM each of folic acid and D-Ca pantothenate, 1 μM riboflavin, and 0.2μM vitamin B12.
 18. A composition comprising neural cells or tissueplaced in the medium of claim
 1. 19. The composition of claim 18 that issupplemented with a serum-free growth promoting supplement thatcomprises effective amounts of hormones, essential fatty acids andanti-oxidants.
 20. The composition of claim 19 wherein the growthpromoting supplement contains biotin, L-carnitine, corticosterone,ethanolamine, D(+)-galactose, reduced glutathione, linoleic acid,linolenic acid, progesterone, putrescine, retinyl acetate, selenium,triodo-1-thyronine, DL-α tocopherol, DL-α tocopherol acetate, bovinealbumin, catalase, insulin, superoxide dismutase and transferrin.
 21. Aholding mixture comprising neural cells or tissue in the medium of claim1.
 22. A process of extending neural cell or tissue viability in anatmosphere having a carbon dioxide concentration of less than about0.25% comprising placing the neural cells or tissue in the medium ofclaim 1 and maintaining the cells or tissue in the atmosphere.
 23. Theprocess of claim 22 wherein the medium is supplemented with a serum-freegrowth promoting supplement that comprises effective amounts ofhormones, essential fatty acids and anti-oxidants.
 24. The process ofclaim 23 wherein the growth-promoting supplement contains biotin,L-carnitine, corticosterone, ethanolamine, D(+)-galactose, reducedglutathione, linoleic acid, linolenic acid, progesterone, putrescine,retinyl acetate, selenium, triodo-1-thyronine, DL-α tocopherol, DL-αtocopherol acetate, bovine albumin, catalase, insulin, superoxidedismutase and transferrin.